Botulinum toxin prefilled container

ABSTRACT

The present invention relates to a prefilled glass container, such as a prefilled glass syringe, comprising an aqueous botulinum toxin formulation. The aqueous botulinum toxin formulation in the prefilled container is stable at low to ambient temperature for a prolonged time period. Furthermore, the present invention relates to a kit comprising the botulinum toxin prefilled container, and to the use of the botulinum toxin prefilled container in therapeutic and cosmetic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/718,236, filed 18 Dec. 2019, which is a continuation of U.S. patentapplication Ser. No. 15/536,503, filed 15 Jun. 2017, now U.S. Pat. No.10,549,042, issued on 4 Feb. 2020, which is a § 371 National StageApplication of PCT/EP2015/002602, filed 22 Dec. 2015, which claimspriority to EP 14004394.4 filed 23 Dec. 2014. Each of these applicationsis incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a prefilled glass container, such as aprefilled glass syringe, comprising an aqueous botulinum toxinformulation. The aqueous botulinum toxin formulation in the prefilledcontainer is stable at low to ambient temperature for a prolonged timeperiod. Furthermore, the present invention relates to a kit comprisingthe botulinum toxin prefilled container, and to the use of the botulinumtoxin prefilled container in therapeutic and cosmetic applications.

BACKGROUND OF THE INVENTION

The stability of pharmaceutical products is of paramount importance toensure safe and efficacious use for a sufficiently long time period.Unfortunately, the performance (safety, reliability, and efficacy) ofmost pharmaceutical products deteriorates over time. The causes of drugdeterioration include chemical degradation (e.g., hydrolysis, oxidation,reduction and racemization), microbial contamination, and othermechanisms (e.g., precipitation).

Proteinaceous active ingredients are often of a labile nature andinherently instable. This leads to loss of biological activity duringproduction, reconstitution and/or storage of protein-containingpharmaceutical compositions. These problems observed with proteins maybe due to chemical instability, resulting in bond formation or cleavage(e.g., hydrolysis, oxidation, racemization, β-elimination and disulfideexchange), and/or due to physical instability of the second orhigher-order structure of proteins without covalent bond-breakingmodification (e.g., denaturation, adsorption to surfaces, andnon-covalent self-aggregation).

Since degradation reactions are generally fastest in aqueous solutionsand slowest in solid dosage forms, protein active ingredients are oftenformulated as lyophilized (i.e. freeze-dried) products. However, thelyophilized products have generally to be reconstituted with apharmaceutically acceptable liquid (e.g., saline) prior to use.Therefore, lyophilized pharmaceutical products are considered lessconvenient than other dosage forms. Further, lyophilized products areusually more expensive and time-consuming to manufacture. Moreover,mismanagement can occur during the reconstitution process resulting ininaccurate dosing or sterility issues. All these disadvantageous can beovercome by the use of prefilled syringes. Therefore, prefilled syringeshave been become increasingly popular as drug delivery devices.

However, if proteins are used as active ingredients, the limitedstability of proteins rendered it often impossible for formulationscientists to use a prefilled syringe format. This applies especially tovery dilute aqueous solutions of botulinum toxin (botulinum neurotoxin,BoNT). Such BoNT solutions are used in the treatment of a wide range ofdebilitating neuromuscular diseases (e.g., cervical dystonia,blepharospasm, spasticity, and hyperhidrosis) and in aesthetic medicine(e.g., treatment of facial wrinkles). There are seven homologousserotypes (A-G) of botulinum toxin, which are produced by differentClostridium spp., in particular C. botulinum, in the form of a complexconsisting of a neurotoxic polypeptide and other (non-toxic) clostridialproteins (i.e. different hemagglutinins and a nontoxic,non-hemagglutinating protein). The neurotoxic polypeptide has amolecular weight of about 150 kDa and is activated by selectiveproteolytic cleavage to yield the active two-chain form consisting of aheavy chain (HC; includes the translocation domain and receptor-bindingdomain) and a light chain (LC; includes the catalytic domain) linked bya disulfide bond and non-covalent interactions.

Botulinum toxins are inherently instable and, in particular, are knownto be highly unstable at alkaline pH and heat-labile. Additionally, itis known that dilution of the isolated toxin complex from milligramquantities to the much lower toxin concentrations used in solutions forinjection (in the nanograms per milliliter range) presents significantdifficulties because of the rapid loss of specific activity upon suchgreat dilution.

Therefore, commercial preparations of botulinum toxin often come asvacuum-dried or lyophilized material. Examples include, for example,Botox® (onabotulinumtoxinA; Allergan, Inc.) and Dysport®(abobotulinumtoxinA; Ipsen Ltd.), which both contain the C. botulinumtoxin complex of type A. Another example is Xeomin® (incobotulinumtoxin;Merz Pharma GmbH & Co. KGaA), which contains the pure neurotoxiccomponent of serotype A (i.e. the neurotoxic polypeptide of a molecularweight of about 150 kDa) and is devoid of any other proteins of theClostridium botulinum toxin complex (i.e. the different hemagglutininsand the nontoxic, non-hemagglutinating protein).

However, the lyophilized toxin products have a number of drawbacksincluding the need for reconstitution prior to use and concomitantsterility issues. In addition, the reconstituted toxin solution is oftennot entirely used in the clinical practice because not every patient andindication requires the same dosage. The unused amount of thereconstituted toxin solution can be stored at lower temperatures, butonly for a short period of time. For example, after dilution with normalsaline prior to use, Botox® and Dysport® are recommended to be usedwithin 6 hours and 4 hours, respectively. Likewise, the package leafletof Xeomin® specifies that after storage for more than 24 hours, thereconstituted Xeomin® solution shall no longer be used than for 24 hoursand is then to be discarded.

To increase toxin stability, stabilizing proteins such as human serumalbumin (HSA) are often added in the art. Other stabilizing strategiesinvolve the use of non-proteinaceous stabilizing agents, for examplesurfactants, polyvinylpyrrolidone (PVP), disaccharides, polyols and thelike. In addition, it was disclosed in WO 00/15245 that a liquidformulation of highly concentrated botulinum toxin type B (about 2500U/ml) is stable for up to 30 months when stored in glass vials at 5° C.However, this prolonged stability requires buffering the pH of theformulation down to an acidic pH of between 5 and 6, which causes painupon injection. Other known approaches to increase toxin stability relyon the addition of various non-protein excipients which, however, areunsuitable or undesirable for human use (see, e.g., WO 01/58472, WO2006/005910, and WO 2007/041664).

Thus, there is still no injectable botulinum toxin presentationavailable which is not only stable over a long period to provide asufficiently long shelf life, but is also convenient and easy to use,reduces medication errors, and minimizes the risk of contamination.

OBJECTIVE OF THE INVENTION

In view of the above, the objective of the present invention is toprovide a medical dosage form for the administration of botulinum toxin,which has a long shelf life and is convenient, safe and easy to use.

SUMMARY OF THE INVENTION

The above object is solved by the provision of a botulinum toxinprefilled container (e.g., a syringe, vial, carpule, or ampoule) whichis characterized by a superior long-term stability of the liquidbotulinum toxin formulation in the container.

In a first aspect, the present invention provides a prefilled glasscontainer (e.g., a syringe, vial, carpule, or ampoule) comprising anaqueous botulinum toxin formulation, wherein the toxin activity is notreduced by more than 25%, preferably by not more than 20%, relative tothe initial toxin activity, upon storage of the prefilled container(e.g., a syringe, vial, carpule, or ampoule) for (a) 12 months at 5° C.,(b) 12 months at 25° C., or (c) 6 months at 30° C.

The stability of the aqueous botulinum toxin formulation in theprefilled container (e.g., a syringe, vial, carpule, or ampoule) interms of the count of subvisible particles equal to or greater than 10μm is also excellent and generally below 1000/ml during storage for 6 to24 months (e.g., 6, 9, 12, 15, 18 or 24 months) at 2-30° C. (e.g., at 5°C., 25° C. or 30° C.). Furthermore, the aqueous botulinum toxinformulation exhibits an excellent pH stability as indicated by a pHvalue that is generally not increased or decreased by more than 10%,relative to the initial pH value, during storage of the prefilledcontainer (e.g., a syringe, vial, carpule, or ampoule) for 6 to 24months (e.g., 6, 9, 12, 15, 18 or 24 months) at 2-30° C. (e.g., at 5°C., 25° C. or 30° C.).

In another aspect, the present invention provides a kit comprising aprefilled glass container (e.g., a syringe, vial, carpule, or ampoule)according to the first aspect of the invention and, optionally,instructions for use of said prefilled glass container.

In a further aspect, the present invention provides a prefilled glasscontainer (e.g., a syringe, vial, carpule, or ampoule) according to thefirst aspect of the present invention for use in therapy. For example,the prefilled glass container (e.g., a syringe, vial, carpule, orampoule) may be used for treating a disease or condition caused by orassociated with hyperactive cholinergic innervation of muscles orexocrine glands in a patient including, but not limited to, dystonia,spasticity, paratonia, diskinesia, focal spasm, strabismus, tremor,tics, migraine, sialorrhea and hyperhidrosis.

In still another aspect, the present invention relates to the use of theprefilled glass container (e.g., a syringe, vial, carpule, or ampoule)according to the first aspect of the invention for use in cosmetictreatments, such as for treating wrinkles of the skin and facialasymmetries, e.g. glabellar frown line, crow's feet, upper facialrhytides and platysma bands.

In a yet further aspect, the present invention provides a method fortreating a disease or condition caused by or associated with hyperactivecholinergic innervation of muscles or exocrine glands in a patient, themethod comprising locally administering an effective amount of botulinumtoxin to a muscle or exocrine gland of the patient using the prefilledcontainer (e.g., a syringe, vial, carpule, or ampoule) according to thefirst aspect of the invention.

In a still further aspect, the present invention relates to a method forthe cosmetic treatment of the skin, such as for treating wrinkles of theskin and facial asymmetries, the method comprising locally administeringan effective amount of botulinum toxin to a patient by intradermal,subdermal or subcutaneous injection using the prefilled glass container(e.g., a syringe, vial, carpule, or ampoule) according to the firstaspect of the present invention.

Further embodiments of the present invention are set forth in theappended dependent claims. The present invention may be more fullyunderstood by reference to the following detailed description of theinvention, the examples and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of a liquid botulinum toxin formulation inprefilled syringe configurations A, B, G, and H at 5° C. as a functionof time. Configuration A: (●), configuration B (▪), configuration H:(□), configuration G: (◯).

FIG. 2 shows the stability of a liquid botulinum toxin formulation inprefilled syringe configurations A, B, G, and H at 25° C. as a functionof time. Configuration A: (●), configuration B (▪), configuration H:(□), configuration G: (◯).

FIG. 3 shows the stability of a liquid botulinum toxin formulation inprefilled syringe configurations A, B, G, and H at 30° C. as a functionof time. Configuration A: (●), configuration B (▪), configuration H:(□), configuration G: (◯).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that a liquidbotulinum toxin formulation in a glass container (e.g., in the form of asyringe, vial, carpule or ampoule) is stable after storage for aprolonged period of time at reduced temperature (e.g., 2-8° C.) and evenat ambient temperature (e.g., 20-30° C., in particular 25° C.). Thebotulinum toxin prefilled container of the present invention thereforeadvantageously exhibits an extended shelf life.

Moreover, the high long-term stability provides tolerance againstinterruptions of the cool chain and may facilitate the approvalprocedure and/or the commercialization in all climate zones, includingcountries with hot climate. Furthermore, the prefilled glass containerof the present invention, in particular the syringe format, presentsseveral additional advantages in comparison to other administrationforms, such as easy and convenient use, reduced risk of medicationerrors, high dosing accuracy, low risk of contamination, improvedsterility assurance, and/or high safety in administration.

As used herein, a “prefilled container” refers to any device having apartially or fully enclosed space that can be sealed or is sealed andcan be used to contain, store, and/or transport liquid formulations. A“prefilled container” within the meaning of the present invention ispreferably a closed (or sealed) container made of, or partially orpredominantly made of, glass and includes, for example, containers inthe form of (i) a syringe, (ii) a vial, (iii) a carpule, or (iv) anampoule.

Prefilled syringes and capsules have two openings that are sealed toprevent leakage of the contents (e.g., aqueous formulations). In case ofa prefilled syringe, the proximal end is sealed by a plunger stopper andthe distal end is sealed by a capping device, as explained in detailherein below. In case of a glass carpule, which is generally a glasscylinder sterile filled with a drug formulation, the proximal end istypically sealed by a rubber stopper. This rubber stopper can be pressedin as a piston in the cylinder by the pressure of a punch of the carpulesyringe. The distal end is typically sealed by a puncture membrane. Thepuncture membrane is pierced for injection.

A “vial” within the meaning of the present invention is a vessel, whichhas usually a tubular form or a bottle-like shape with a neck and issuitable for containing, storing, and/or transporting drug formulations.The single opening is sealable by different vial closure systems. Forexample, vials may be closed with a screw cap (screw vials), a stopperof cork, plastic or rubber (lip vials and crimp vials) and other closuresystems like flip-tops or snap caps. Within the present invention a“vial” preferably means a glass vessel having its opening sealed with avial closure system.

In the following, the present invention is described in more detail. Itis pointed out that, although the term “prefilled syringe”, “prefilledglass syringe”, “syringe” or “glass syringe” is used in the detaileddescription of the invention, this does not mean that it is limited to a(glass) syringe as a particular embodiment of the (glass) container. Infact, any reference herein to a “prefilled syringe”, “prefilled glasssyringe”, “syringe”, “glass syringe” or the like is to be understood asa reference to, and disclosure of, a “container” or “glass container”and also includes, or discloses, a “vial” or “glass vial”, a “carpule”or “glass carpule”, or an “ampoule” or “glass ampoule”, unless otherwisestated.

In a first aspect, the present invention relates to a prefilled glasssyringe comprising botulinum toxin in an aqueous formulation, whereinthe toxin activity is not reduced by more than 25%, relative to theinitial toxin activity, upon storage of the prefilled syringe for (a) 12months at standard refrigerator temperatures (i.e. 2-8° C., such as 5°C.), (b) 12 months at 25° C., or (c) 6 months at 30° C. Preferably, thetoxin activity is not reduced by more than 20% or 15%, relative to theinitial toxin activity, upon storage of the prefilled syringe for (a) 12months at 2-8° C. (e.g., 5° C.), (b) 12 months at 25° C., or (c) 6months at 30° C. More preferably, the toxin activity is not reduced bymore than 20% or 15%, relative to the initial toxin activity, uponstorage of the prefilled syringe for (a) 6 months at 2-8° C. (e.g., 5°C.), (b) 6 months at 25° C., or (c) 3 months at 30° C. Particularlypreferable, the toxin activity is not reduced by more than 10%, relativeto the initial toxin activity, upon storage of the prefilled syringe for(a) 3 to 6 months at 2-8° C. (e.g., 5° C.) or (b) 3 to 6 months at 25°C. Especially preferable, the toxin activity is not reduced by more than5%, relative to the initial toxin activity, upon storage of theprefilled syringe for (a) 3 to 6 months at 2-8° C. (e.g., 5° C.) or (b)3 to 6 months at 25° C.

Surprisingly, the aqueous botulinum toxin formulation in the prefilledsyringe is also stable for even longer storage times of up to 24 monthsor even longer. For example, upon storage for up to 24 months (e.g., 15,18 or 24 months) at 2-8° C. (e.g., 5° C.) or 25° C., the toxin activityis preferably not reduced by more than 30% or 25%, more preferably by nomore than 20%, in particular by no more than 15%, particularlypreferable by no more than 10%, and most preferable by no more than 5%,relative to the initial toxin activity.

In particular, the toxin activity is preferably not reduced by more than25%, 20%, 15%, 10% or 5%, relative to the initial toxin activity, uponstorage of the prefilled syringe for 24 months at 2-8°. Upon storage ofthe prefilled syringe at 2-8° for 18 months, the toxin activity ispreferably not reduced by more than 25%, 20%, 15%, 10% or 5%, relativeto the initial toxin activity. Furthermore, the toxin activity ispreferably not reduced by more than 35%, 30%, 25%, 20% or 15%, relativeto the initial toxin activity, upon storage of the prefilled syringe for24 months at 25° C. Upon storage of the prefilled syringe at 25° C. for18 months, the toxin activity is preferably not reduced by more than30%, 25%, 20%, 15% or 10%, relative to the initial toxin activity.

Within the present invention, the term “toxin activity” is intended torefer to the biological activity of the botulinum toxin. “Biologicalactivity” may refer to (a) receptor binding, (b) internalization, (c)translocation across the endosomal membrane into the cytosol, and/or (d)endoproteolytic cleavage of proteins involved in synaptic vesiclemembrane fusion. For example, any LC (ligh chain) domain, which showsproteolytic activity of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% and up to 100% of the corresponding wild-type LC domain in aSNAP-25 assay may be considered “biological active” or “to exhibitproteolytic activity” within the scope of this invention. Furthermore,any HC (heavy chain) domain that is capable of binding to a cellular HCdomain receptor, in particular to its native HC domain receptor, and iscapable of translocating an LC domain attached to it, is considered“biologically active”.

The biological activity is expressed in Mouse Units (MU). As usedherein, 1 MU is the amount of neurotoxic component, which kills 50% of aspecified mouse population after intraperitoneal injection, i.e. themouse i.p. LD₅₀, as measured in accordance with the method of Schantzand Kauter (Schantz and Kauter, J. Assoc. Off. Anal. Chem. 1978,61:96-99). The terms “MU” and “Unit” or “U” are used interchangeableherein.

Suitable assays for assessing the biological activity include the mousehemidiaphragm assay (MHA) described by Pearce et al. (Toxicol. Appl.Pharmacol. 128:69-77, 1994), the hemidiaphragm assay (HDA) according toGoschel et al. (Experimental Neurology 147:96-102, 1997), the mousediaphragm assay (MDA) according to Dressler et al. (Mov. Disord.20:1617-1619, 2005), the SNAP-25 protease assay (e.g., the “GFP-SNAP25fluorescence release assay” described in WO 2006/020748 or the “improvedSNAP25 endopeptidase immuno-assay” described in Jones et al., 2008, J.Immunol. Methods 329:92-101), the electrochemoluminescence (ECL)sandwich ELISA described in WO 2009/114748, and cell-based assays asthose described in WO 2009/114748, WO 2004/029576, WO 2013/049508 and,in particular, WO 2014/207109.

As used herein, the term “initial toxin activity” or “initial potency”generally refers to the activity of the botulinum toxin at the beginningof the storage period, i.e. after manufacture of the final, sterilizedbotulinum toxin prefilled syringe, in particular directly aftermanufacture or within one or two days after manufacture. Further, theterm “upon storage”, as used herein is intended to mean after storagefor a certain time period. In addition, the term “during storage”generally means over the course of the entire storage period.

Furthermore, the aqueous botulinum toxin formulation is highly stable interms of the subvisible particle count. A “subvisible particle” withinthe meaning of the present invention is typically a particle with adiameter below 100 μm. Specifically, the count (or number) of particlesequal to or greater than 10 μm in the aqueous botulinum toxinformulation is typically below 1000/ml, preferably below 600/ml and morepreferably below 200/ml during storage for 6 to 24 months (e.g., 6, 9,12, 15, 18 or 24 months) at 2-30° C. (e.g., at 5° C., 25° C. or 30° C.).

Particle measurements may be conducted by different methods, such asMicro-Flow Imaging (MFI), Resonant Mass Measurement (RMM), andNanoparticle Tracking Analysis (NTA). The particle measurements usuallyfollow USP <788>. Within the context of the present invention, theMicro-Flow Imaging measurement is preferably used. This measurementmethod may, for example, be conducted using a DPA-5200 particle analyzersystem (ProteinSimple, Santa Clara, Calif., USA) equipped with a silanecoated high-resolution 100 μm flow cell. Generally, the samples areanalyzed undiluted.

Alternatively, Resonant Mass Measurements (RMM) may be employed todetermine the number of particles using, for example, the ARCHIMEDESParticle Metrology System (Affinity Biosensors, Santa Barbara, Calif.,USA) equipped with a microsensor (size range 0.3-4 μm) calibrated with 1μm polystyrene standards. All samples are typically analyzed withoutdilution. The results may be analyzed using the ParticleLab software(v1.8.570) with a size bin step of 10 nm. As another alternative fordetermining the particle count, Nanoparticle Tracking Analysis (NTA) maybe used, for example, using a NanoSight LM20 system (NanoSight,Amesbury, UK). The samples are typically measured undiluted. Movementsof the particles in the samples may be recorded as videos for 60 secondsat ambient temperature and analyzed using suitable software (e.g., theNTA 2.3 Software).

Moreover, the aqueous botulinum toxin formulation shows high pHstability in that the pH value is essentially stable during storage ofthe prefilled syringe. Preferably, the pH value is not increased ordecreased by more than 10%, 8% or 6%, relative to the initial pH value,upon storage of the prefilled syringe for 6 to 24 months (e.g., 6, 9,12, 15, 18 or 24 months) at 2-30° C. (e.g., at 5° C., 25° C. or 30° C.),for example for 18 months at 25° C. or for 24 months at 25° C. The pHmay be measured in accordance with the US Pharmacopeia standardized testmethod USP <791>, which outlines pH measurements for a multitude ofpharmaceutical product. Any suitable pH meter may be used, for examplethe Lab 870 pH meter of Schott Instruments.

As used herein, the term “prefilled syringe” refers to a syringe whichis filled with a drug composition (i.e. the aqueous botulinum toxinformulation) prior to distribution to the end user who will administerthe drug to the patient. A prefilled syringe typically includes a drugcontainment container forming part of a syringe body (i.e. a syringebarrel), a plunger to seal the proximal opening of the syringe and forexpelling the drug, and a sealing device (e.g., a tip cap or a needleshield) on the outlet end of the syringe (e.g., the open end of thesyringe tip or of a pre-mounted needle (cannula)) to seal the distaloutlet opening. The term “prefilled glass syringe”, as used herein,refers to a prefilled syringe, of which at least the barrel is made ofglass.

Within the present invention, the prefilled syringe is preferably a Luerslip or Luer lock syringe equipped with a tip cap (if no needle ispre-mounted) or a needle shield (if the needle is pre-mounted). Withinthe meaning of the present invention, a “luer slip syringe” is a syringethat allows a needle to be pushed on to the end of the tip, whereas a“Luer-Lock syringe” is a syringe that allows a needle to be twisted ontothe tip and then locked in place. This provides a secure connection andprevents accidental removal of the needles of the injection of fluids.

The prefilled syringe according to the present invention is generallysterilized and, thus, ready-to-use. Further, the prefilled syringedescribed herein is usually intended for single use and intended to bedisposable. Prior to sterilization, the syringe, more specifically theinner surface of the glass syringe barrel, is typically coated with alubricant to ease gliding of the plunger stopper and extruding of thesyringe content. Suitable methods for sterilization include, but are notlimited to, gamma radiation, ethylene oxide (ETO) treatment and moistheat (e.g., autoclaving).

In accordance with the present invention, the aqueous botulinum toxinformulation in the prefilled syringe contains the botulinum toxin at aconcentration of, for example, 1 U/ml to 3000 U/ml, 10 U/ml to 1000U/ml. Preferably, the botulinum toxin is present at a concentration ofabout 10 U/ml to 400 U/ml, more preferably about 25 U/ml to 200 U/ml,and most preferably about 40 U/ml to 150 U/ml (e.g., 50 U/ml, 75 U/ml or100 U/ml).

The term “botulinum toxin”, as used herein, broadly refers to any formand type of botulinum toxin. In particular, the botulinum toxin may beselected from botulinum toxin types A, B, C1, D, E, F, G, or mixturesthereof. Within the context of the present invention, the botulinumtoxin is preferably of serotype A, B or C1, in particular serotype A.

Furthermore, the term “botulinum toxin”, as used herein, is intended toinclude both the botulinum toxin complex (the “toxin complex”) and the“neurotoxic component” of a botulinum toxin complex. As used herein, theterm “botulinum toxin complex” or “toxin complex” refers to a highmolecular weight complex comprising the neurotoxic component ofapproximately 150 kDa and, in addition, non-toxic proteins ofClostridium botulinum, including hemagglutinin and non-hemagglutininproteins. The botulinum toxin serotype A complex is commerciallyavailable, for example, as Botox® (Allergan, Inc.) or as Dysport®(Ipsen, Ltd.).

The term “neurotoxic component”, as used herein, relates to theneurotoxic polypeptide of the toxin complex (the “150 kDa” polypeptide)without any associated non-toxic proteins. The pure neurotoxic componentis, for example, commercially available under the trade names Xeomin®and Bocouture® (Merz Pharmaceuticals GmbH). Within the presentinvention, the botulinum toxin is preferably the neurotoxic component ofa botulinum toxin complex of, e.g., serotype A, B, C1, in particular ofa botulinum toxin complex of serotype A. In other words, the aqueousbotulinum toxin formulation contained in the prefilled glass syringepreferably contains (only) said neurotoxic component and is devoid ofany other protein of the Clostridium botulinum toxin complex.

It is also contemplated that the present invention encompasses isoforms,homologs, orthologs and paralogs of botulinum toxin that show at least50%, at least 60%, at least 70%, at least 80%, at least 90% and up to60%, up to 70%, up to 80%, up to 90%, up to 100% sequence identity towild-type botulinum toxin, e.g. wild-type botulinum toxin A or theneurotoxic component of botulinum toxin of serotype A1 deposited withthe GenBank database under the accession number AAA23262. The sequenceidentity can be calculated by any algorithm suitable to yield reliableresults, for example by using the FASTA algorithm (W. R. Pearson & D. J.Lipman PNAS (1988) 85:2444-2448). Sequence identity may be calculated bycomparing two polypeptides or two domains such as two LC domains orfragments thereof.

Modified and recombinant botulinum toxins are also within the scope ofthe present invention. With respect to suitable mutants, reference ismade to WO 2006/027207, WO 2009/015840, WO 2006/114308, WO 2007/104567,WO 2010/022979, WO 2011/000929 and WO 2013/068476, which are allincorporated by reference herein. Furthermore, the present inventionalso refers to botulinum toxins, which are chemically modified, e.g. bypegylation, glycosylation, sulfatation, phosphorylation or any othermodification, in particular of one or more surface or solvent exposedamino acid(s). The modified, recombinant, isoforms, homologs, orthologs,paralogs and mutants suitable for use in the present invention arebiologically active, i.e. able to translocate into neurons and cleaveproteins of the SNARE complex (e.g., VAMP/syntaxin, synaptobrevin, andSNAP-25) to exert its acetylcholine inhibitory effects, e.g., its muscleparalyzing effects.

Within the context of the present invention, the aqueous botulinum toxinformulation may comprise various other pharmaceutically acceptablesubstances, for example, salts (e.g., sodium chloride), stabilizingproteins (e.g., albumin, gelatin), sugars (e.g., glucose, fructose,galactose, trehalose, sucrose and maltose), carbohydrate polymers (e.g.,hyaluronic acid and polyvinylpyrollidone (PVP)), polyols (e.g. glyceroland sugar alcohols like mannitol, inositol, lactilol, isomalt, xylitol,erythritol, sorbitol), amino acids, vitamins (e.g. vitamin C), zinc,magnesium, anesthetic agents (e.g., local anesthetic agents likelidocaine), surfactants, tonicity modifiers, and the like. The term“pharmaceutically acceptable”, as used herein, refers to those compoundsor substances which are suitable for contact with the tissues ofmammals, especially humans.

The term “comprise”, as used herein, is intended to encompass both theopen-ended term “include” and the closed term “consist (of)”. The term“made of”, as used herein, is intended to broadly relate to “producedof/from”, in particular mainly produced from, and generally means“comprising” (indicating that other substances or materials may beincluded in some amounts). It may also mean “consisting of”.

Preferably, the pH of the aqueous botulinum toxin formulation in theprefilled syringe is between 6.0 to 7.5, 6.5 to 7.5, 6.1 to 7.3, 6.2 to7.2, 6.3 to 7.1, and 6.5 to 7.0 during storage. A pH within the range of6.1 to 7.3 is advantageous in that injections of such neutral or onlyslightly acidic solutions are much less painful upon injection thanacidic solutions.

The term “aqueous formulation” or “aqueous botulinum toxin formulation”,as used herein, is not particularly limited and may refer to an aqueoussuspension, aqueous dispersion, aqueous emulsion and is preferably anaqueous solution.

Preferably, the aqueous botulinum toxin formulation does not contain abuffer substance like a phosphate buffer, a phosphate-citrate buffer, alactate buffer, an acetate buffer and the like. The term “buffer” asused herein denotes a pharmaceutically acceptable excipient, whichstabilizes the pH of a pharmaceutical preparation. Furthermore, theaqueous botulinum toxin formulation may be free of amino acids (e.g.,methionine) and/or surfactants (e.g., polysorbates such as polysorbate80) and/or animal-derived proteins (e.g., human serum albumin (HSA) orbovine serum albumin (BSA)).

A preferred aqueous botulinum toxin formulation for use herein compriseswater, botulinum toxin (e.g., the neurotoxic component of botulinumtoxin, preferably of type A) at a concentration such as 10 to 150 U/ml,a salt (e.g., sodium chloride) in a concentration such as 0.5% to 1.5%w/v, a sugar (e.g., a mono- or disaccharide, such as glucose, fructose,galactose, trehalose, sucrose and maltose) at a concentration such as0.1% to 2% w/v, and a stabilizing protein (e.g., albumin) at aconcentration such as 0.01% to 4% w/v, 0.1% to 3% w/v, or 0.1% to 1%w/v.

Another preferred aqueous botulinum formulation for use hereinessentially consists of water, botulinum toxin (e.g. the neurotoxiccomponent of botulinum toxin type A), sodium chloride, sucrose, andalbumin (e.g., human serum albumin; HSA). The concentration of thementioned components may be in the following ranges: 10 to 200 U/ml or30 to 125 U/ml (botulinum toxin), 0.5% to 1.5% w/v or 0.7% to 1.1% w/v(sodium chloride), 0.1% to 2% w/v or 0.2% to 1% w/v (sucrose), 0.01% to1% w/v, 0.05% to 0.5% w/v, 0.1% to 3% w/v or 0.5% to 1.5% w/v (HSA). Afurther preferred botulinum toxin formulation for use herein is aXeomin® solution, e.g., reconstituted with physiological saline (0.9%sodium chloride), including 20 to 150 U/ml of the neurotoxic componentof botulinum toxin type A.

The term “essentially consists of”, as used herein is intended to meanthat substances other than those indicated are only contained in traceamounts, e.g. unavoidable impurities contained in the components usedfor formulating the aqueous botulinum toxin formulation, and low amountsof impurities included in the isolated botulinum toxin (e.g., theneurotoxic component of botulinum toxin type A) as a result of thepurification procedure (e.g., very low residual amounts of buffers,chelating agents and the like).

In accordance with the present invention, the configuration of theprefilled syringe is not particularly limited and commonly comprises afluid-receiving barrel that, after filling, is removably capped by acapping device to sealingly close the distal end of the syringe (e.g.,by a “cap” (or “tip cap”) that is removed and replaced by a needle priorto use, or a sealing means like a needle shield in case of a syringewith a removable or permanent needle), and is closed at the proximal endby its plunger or any other means that is in fluid-tight engagement withthe inner wall of the barrel. To use the prefilled syringe, the tip cap,needle shield or other type of capping device are removed, optionally aneedle is attached (if not already present), and the plunger tip orpiston is advanced in the barrel to inject the contents of the barrelinto a patient.

The prefilled glass syringe according to the present inventionpreferably comprises:

-   -   (a) a syringe barrel made of glass including a proximal end and        a distal end, and a generally cylindrical wall extending        therebetween and defining a barrel lumen, the syringe barrel        having a distally projecting tip with a fluid passage extending        therethrough and communicating with the barrel lumen, wherein        the generally cylindrical wall has an interior surface        optionally coated with a barrier layer,    -   (b) a capping device having an outlet engaging portion sealingly        engaging and closing the distal open outlet end of the syringe,        wherein the outlet engaging portion is made of an elastomeric        material that optionally has a coating on its surface, and    -   (c) a plunger rod assembly which extends into the proximal end        of the syringe barrel and includes a plunger stopper in sliding        fluid-tight engagement with the cylindrical wall of the barrel        lumen, wherein the plunger stopper is made of an elastomeric        material, which optionally has a coating on at least a portion        of the plunger stopper that contacts the aqueous botulinum toxin        formulation during storage and/or injection.

Generally, primary container closure systems, including components suchas syringe barrels, sealing devices (e.g., tip caps or needle shields)and plungers, have the potential to interact with the drug formulationin the prefilled syringe. This may lead to the release ofextractables/leachables from the syringe materials that contact theaqueous botulinum toxin formulation. The extractables/leachables havethe potential to contaminate the aqueous botulinum toxin formulation andto impair the stability or activity of the botulinum toxin. Therefore,the materials of the prefilled syringe are generally selected tominimize or limit the amount of extractables and leachables.

As used herein, the terms “extractable(s)” and “leachable(s)” refer tochemical species that can be released from a container or component ofmaterial of the prefilled syringe and/or has migrated from syringematerials into the aqueous botulinum toxin formulation under normalconditions of use or storage. Methods for identification ofextractables/leachables are known in the art and based on recommendedindustry practices and International Conference for Harmonisation (ICH)guidelines (see FDA guidance, Container Closure Systems for PackagingHuman Drugs and Biologics), and include, e.g., LiquidChromatography/Mass Spectrophotometry (LC/MS), Gas ChromatographySpectroscopy/Mass Spectrophotometry (GC/MS), Inductively Coupled Plasma(ICP) and Infrared (IR).

The inside surface of the glass barrel is usually coated with alubricant layer (herein also referred to, and interchangeably used with,the term “barrier layer” or “barrier coating”). The lubricant layershould not only provide high lubricity, enabling the plunger to easilyglide through the barrel, but also be compatible with the aqueousbotulinum toxin formulation and protect its shelf life. Within thecontext of the present invention, the lubricant layer may be asilicone-free lubricant layer or a silicone lubricant layer.

Likewise, the inner surface of the glass vessel part of the vial, theinner surface of the glass cylinder of the carpule, and the innersurface of the glass ampoule may be optionally coated with a barrierlayer and, in particular, with a silicone-free layer or a siliconelayer. Thus, all comments provided below with regard to thesilicone-free lubricant layer and the silicone lubricant layer of theglass syringe equally apply to the silicone-free layer and siliconelayer, respectively, of the glass vial, glass carpule and glass ampoule.

A suitable silicone-free lubrication layer is, for example, afluoropolymer layer (e.g., fluoropolymer (fluorocarbon) layers, such asethylene-tetrafluoroethylene (ETFE) layers and perfluoropolyether-based(PFPE-based) layers (e.g., TriboGlide®)), as well as silicon oxide-basedglass PECVD (plasma-enhanced chemical vapor deposition) coatings.

The silicone-free lubrication layer can be prepared as known in the art,for example by spraying glass syringe barrels with a perfluoropolyetheroil to achieve a thin layer of lubricant on the inside surface of thesyringe, followed by exposing the inner cavities to a downstream inertgas (e.g., argon or helium) plasma. The plasma treatment leads tocrosslinking of the perfluoropolyether, thereby immobilizing the coatingand reducing its tendency to migrate off the target surface, resultingin less particles that potentially impairs the stability/efficacy of thebotulinum toxin drug. An exemplary production process is described in WO2014/014641 A1, the content of which is incorporated herein byreference. Furthermore, a particularly suitable silicone-free barriercoating for use herein is known in the art as TriboGlide® coating, aperfluoropolyether coating crosslinked by plasma treatment.

A suitable silicone lubricant layer for use herein may be prepared by asiliconization method selected from, but not limited to, siliconeoil-based methods (e.g., spray-on siliconization or baked-onsiliconization) and vapor deposition methods (e.g., plasma enhancedchemical vapor deposition (PECVD)). Preferably, the silicone lubricantlayer is formed by spray-on siliconization or, more preferably, bybaked-on siliconization.

In the spray-on siliconization method, a silicone oil (e.g. DOW CORNING®360 with a viscosity of 1000 cSt) is sprayed into the syringe (i.e. thebarrel) using, e.g., a diving or static nozzle to produce a thinsilicone oil layer. While silicone oil is an excellent lubricant, excesssilicone oil can lead to the formation of unwanted visual and subvisualsilicone oil particles. With protein-based drugs, in particular, thesesilicone oil particles may lead to undesirable interactions with proteindrugs. For example, subvisual silicone oil particles are thought topromote protein aggregation. Therefore, since it results in fewersub-visual and visual silicone oil particles, the baked-onsiliconization processes is particularly preferred for use herein. Itinvolves the application of silicone oil as an emulsion (e.g., DOWCORNING® 365 siliconization emulsion), which is then baked on the glasssurface at a specific temperature and for a specific time.

The design of the syringe barrel is not particularly limited andtypically has an inside diameter adjusted to accommodate the desiredfill volume of, e.g., 0.5 cm³, 1.0 cm³, 1.5 cm³ or 2.0 cm³. Usually, thesyringe barrel has graduated marks indicating the volume of fluid in thesyringe. In addition, the syringe barrel may include a flange-styleinterface. The design of the flange may, for example, be compatible withISO11040. The flange-style interface may further be compatible with anoptionally present handle. Furthermore, in case of a Luer-Lock syringe,the syringe may be equipped with a Luer-Lock adaptor of, e.g.,polycarbonate.

The syringe tip is usually integrally formed with the syringe barrel.The tip is formed with an integral passage extending axially through thetip and being in communication with the chamber for dispensing thecontents of the syringe barrel. The tip may have a substantiallyfrustoconical shape that converges from the distal outlet end of thesyringe barrel towards the tip's outlet end. Alternatively, the tip maybe characterized as divergent (i.e., expanding from a smaller diameterto a larger one). Furthermore, the tip is usually located centrally inrelation to the body of the syringe (concentric syringe tip) but mayalso be located offset towards the edge of the body (eccentric syringetip).

The “capping device” within the meaning of the present invention broadlyrefers to any means for closing and sealing the open outlet end of asyringe. Within the present invention, the term “open outlet end” refersto any distal open end of a syringe that is in fluid communication withthe barrel lumen. The capping device generally has a channel with aclosed end and an open end having a dimension for receiving andefficiently sealing the open outlet end of the syringe to preventleakage.

In case of prefilled syringes without pre-mounted needles, the cappingdevice is a capping means commonly known as “tip cap”. The tip cap formsa fluid-tight seal with the tip of the syringe to efficiently close thesyringe barrel and to prevent leakage of the contents of the syringebarrel. The tip cap is usually removable coupled to the syringe tip or aluer collar. The luer collar surrounds the top of the syringe barrel(e.g., syringe tip). Preferably, the luer collar has internal threadsand the tip cap has external threads complementing said internal threadsof said luer collar for coupling the tip cap to the syringe barrel. Theluer collar is typically a separately molded luer collar that is mounteddirectly to the tip of the syringe barrel, e.g., by a snap orinterference fit. Prior to use, the tip can be removed, and a needlecannula (or needle/needle assembly) can then be securely coupled to thesyringe tip.

If the prefilled syringe includes a removable or non-removable(permanent) cannula (also referred to as “needle” or “needle assembly”)extending from the syringe tip for delivering the aqueous botulinumtoxin formulation from said syringe, the capping device may be referredto as “needle shield”. Said needle shield generally has a channel with aclosed end and an open end having a dimension for receiving and couplingwith the cannula (needle) mounted on the tip of the syringe. Typically,the (sharpened) end of the cannula penetrates the closed end of thechannel in the needle shield to seal the open end of the cannula.

The capping device (e.g., tip cap or needle shield) may be a unitarymember and is usually made from a flexible and/or resilient polymericmaterial (e.g., an elastomer), at least a portion of which contacts andseals the distal opening of the syringe (referred to as the “outletengaging portion”). Alternatively, the capping device may have an outercap made of a rigid plastic material that is coupled to a flexibleand/or resilient inner cap made of a flexible and resilient polymericmaterial (e.g., an elastomer), wherein at least a portion of the innercap contacts and seals the distal opening of the syringe (referred to asthe “outlet engaging portion”).

In view of the fact that the outlet engaging portion contacts theaqueous botulinum toxin formulation during storage and/or use, it ispreferably made of a material having a minimized potential for unwantedextractables/leachables. To this end, the outlet engaging portion mayhave a coating thereon to increase compatibility with the aqueousbotulinum toxin formulation.

Suitable flexible and/or resilient materials of the capping device, inparticular of the outlet engaging portion, include elastomers that donot interfere with the aqueous botulinum toxin formulation and enablelong-term storage. In particular, the part of the sealing device thatcontacts the aqueous botulinum toxin formulation, i.e. the outletengaging portion, should exhibit low extractable/leachable levels duringprolonged storage of the aqueous botulinum toxin formulation. As usedherein, the term “elastomeric” or “elastomeric material” refersprimarily to crosslinked thermosetting rubbery polymers that are moreeasily deformable than plastics but that are approved for use withpharmaceutical grade fluids and are not readily susceptible to leachingor gas migration.

Preferably, the elastomeric material is selected from isoprene rubber(IS), butadiene rubber (polybutadiene, BR), butyl rubber (copolymer ofisobutylene and isoprene; IIR), halogenated butyl rubber (e.g., chlorobutyl rubber, CIIR; and bromo butyl rubber: BIIR), styrene-butadienerubber (copolymer of styrene and butadiene, SBR), and mixtures thereof.Preferably, the elastomeric material is a styrene-butadiene rubber, abutyl rubber, a blend of isoprene rubber and a halogenated (e.g. bromoor chloro) butyl rubber, a halogenated butyl rubber, particularly abromo butyl rubber or a chloro butyl rubber, or a mixture thereof. Theelastomeric material may also be reinforced with an inert mineral.Further, it may be cured (e.g., with organic peroxide, phenolic resins,etc.).

Suitable coatings that may be optionally present on the elastomericmaterial are made of a material that does not undesirably interfere withthe aqueous botulinum toxin formulation and exhibits low levels ofextractables/leachables. A preferred example of such a coating is acoating made of a fluoropolymer, i.e. a fluorocarbon coating. Othersuitable coatings for use herein include, for example, polypropylene,polyethylene, parylene (e.g., parylene N, parylene C and parylene HT),and crosslinked silicone (e.g., the the B2-coating (Daikyo Seiko) orXSi™ (Becton Dickinson)).

The fluoropolymer coatings include, but are not limited to, fluorinatedethylene-propylene copolymers (e.g.,tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), fluorinatedethylene-ethylene copolymers (e.g., ethylene tetrafluoroethylenecopolymer (ETFE), such as FluroTec®), PVA (a copolymer oftetrafluoroethylene (TFE) and perfluoropropylvinylether (PPVE)),tetrafluoroethylene-perfluoroethylene copolymers, polyvinylidenefluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene(PTFE), and mixtures thereof. Preferably, the coating is made of ETFEand, particularly, is a FluroTec® coating.

With regard to the carpule of the present invention, the distal end issealed by a puncture membrane. The puncture membrane may be formed froma thin rubber or silicone, a thin plastic/polymer, a film such as Mylar,a polyolefin such as polyethylene or polypropylene, a metal foil such analuminum foil, etc. The membrane may be between about 0.001 and 2.0 mm,usually between 0.002 mm and 0.65 mm thick. Also, the membrane may bemade of an elastomeric material and optionally have a coating asdescribed above in connection with the capping device of the prefilledglass syringe.

With regard to the vial of the present invention, the vial closuresystem (e.g., cap), in particular those portions of the vial closuresystem that come into contact with, or have the potential to come intocontact with and/or seal the vial (e.g., a septum) may be made of anelastomeric material, in particular a thermoplastic elastomericmaterial, more particularly a styrenic block copolymer thermoplasticelastomer, or of an elastomeric material as described above inconnection with the capping device of the prefilled glass syringe of thepresent invention. Another suitable material is a silicone material.Furthermore, the said materials may have an optional coating, inparticular a fluoropolymer coating, as defined above in relation to thecapping device of the prefilled glass syringe.

In accordance with the present invention, the prefilled syringe commonlyincludes a plunger rod assembly, which extends into the proximal end ofthe syringe barrel. The plunger rod assembly may include a rod (alsoknown as pushrod) with a plunger stopper at its tip (also known as“plunger”) in sliding fluid-tight engagement with the cylindrical wallof the barrel lumen. The plunger forms the proximal seal and the dynamicseal that allows for extrusion of the liquid botulinum toxinformulation. The plunger stopper contacts the aqueous botulinum toxinformulation during storage and/or administration. Therefore, the plungerstopper should be compatible with the aqueous botulinum toxinformulation and not impair its long-term stability. In particular, theplunger stopper should preferably be designed to minimize the amount ofextractables/leachables upon long-time storage.

Within the present invention, the plunger stopper is preferably made ofan elastomeric material, which optionally has a coating on at least aportion of the plunger stopper that contacts the aqueous botulinum toxinformulation during storage and/or use. Suitable plunger stopperelastomeric materials for use herein include, but are not limited to,isoprene rubber (IS), butadiene rubber (polybutadiene, BR), butyl rubber(copolymer of isobutylene and isoprene, IIR), halogenated butyl rubber(e.g., chloro butyl rubber, CIIR; and bromo butyl rubber, BIIR),styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), andmixtures thereof. Preferably, the plunger stopper material is a butylrubber or a halogenated butyl rubber or a mixture thereof, morepreferably a bromo butyl rubber or a chloro butyl rubber, and mostpreferably a butyl rubber. The elastomeric material may also bereinforced with an inert mineral. Further, it may be cured (e.g., withorganic peroxide, phenolic resins, etc.).

Preferably, the plunger stopper comprises a coating acting as a barrierfilm. The coating is usually applied to at least the seal surfaces,including the surface portion of the plunger stopper facing the barrellumen and contacting the aqueous botulinum toxin formulation duringstorage and/or use. The coating serves the purpose of minimizinginteraction between the plunger and the liquid botulinum toxinformulation and to provide good lubricity.

Suitable coatings of the plunger stopper are generally made of amaterial that does not undesirably interfere with the aqueous botulinumtoxin formulation and exhibits low levels of extractables/leachables.Such coatings include, but are not limited to, polypropylene,polyethylene, parylene (e.g., parylene N, parylene C and parylene HT),crosslinked silicone and, preferably, fluoropolymer coatings. Examplesof suitable crosslinked silicone coatings include the B2-coating (DaikyoSeiko) or XSi™ (Becton Dickinson).

The fluoropolymer coatings include, but are not limited to, fluorinatedethylene-propylene copolymers (e.g.,tetrafluoroethylene-hexafluoropropylene copolymer (FEP)), fluorinatedethylene-ethylene copolymers (e.g., ethylene tetrafluoroethylenecopolymer (ETFE), such as FluroTec®), PVA (a copolymer oftetrafluoroethylene (TFE) and perfluoropropylvinylether (PPVE)),tetrafluoroethylene-perfluoroethylene copolymers, polyvinylidenefluoride (PVDF), polyvinyl fluoride (PVF), polytetrafluoroethylene(PTFE), and mixtures thereof. Preferably, the coating is made of ETFEand, particularly, is a FluroTec® coating.

The design of the plunger stopper is not particularly limited and may bea nested or bagged stopper. Further, the interface to the rod may bethreaded to allow installation of the rod after sterilization.Alternatively, the interface to the rod may be designed with a snap-ondesign. The rod, like the plunger stopper, is generally designed towithstand sterilization but is not otherwise limited in any particularway. Typically, the rod is made of a plastic material such as anethylene vinyl acetate (EVA) copolymer or a polypropylene.

The rubber stopper of the carpule of the present invention may be madeof the same elastomeric materials as described above in connection withthe plunger stopper of the glass syringe. Also, the rubber stopper ofthe carpule may have the same optional coating as defined above withrespect to the coating on the plunger stopper. Further, the coating maybe on at least a portion of the rubber stopper that contacts the aqueousbotulinum toxin formulation during storage and/or use.

The prefilled syringe of the present invention meets the industrystandard with regard to extractables, such as defined by the foamingtest, pH test, potassium permanganate-reducing substances test, UVspectrum test and residue on evaporation test according to The JapanesePharmacopoeia, No. 61, Test Methods for Plastic Containers (2001).Furthermore, the prefilled syringe and the respective component beforeand after sterilization satisfy the standards of The JapanesePharmacopoeia, 14th Edition, No. 59, Test for Rubber Closure for AqueousInfusions.

In another aspect, the present invention relates to a kit comprising aprefilled glass container (e.g., a syringe, vial, carpule or ampoule)according to the present invention and, optionally, instructions for useof said prefilled glass container.

In a further aspect, the present invention relates to a prefilled glasssyringe according to the present invention for use in therapy. Inparticular, the prefilled glass container (e.g., a syringe, vial,carpule or ampoule) according to the present invention may be used inthe treatment of a disease or condition caused by or associated withhyperactive cholinergic innervation of muscles or exocrine glands in apatient.

Within the context of the present invention, if the container is not asyringe (e.g., a vial, carpule or ampoule), the content of these“non-syringe type” containers (i.e. the aqueous botulinum toxinformulation) is generally injected to the desired target site using asuitable injection device (e.g., a syringe) in the same manner asdescribed herein in relation to the prefilled glass syringe. Thecarpules are inserted into a carpule injection device as known to thoseskilled in the art. The contents of the vials and ampoules are generallyaseptically filled into a syringe and then injected to the target siteusing a suitable injection device (e.g., a syringe) in the same manneras described herein in relation to the prefilled glass syringe.

The term “hyperactive cholinergic innervation”, as used herein, relatesto a synapse, which is characterized by an unusually high amount ofacetylcholine release into the synaptic cleft. “Unusually high” relatesto an increase of, e.g., up to 25%, up to 50% or more with respect to areference activity which may be obtained, for example, by comparing therelease with the release at a synapse of the same type but which is notin a hyperactive state, wherein muscle dystonia may be indicative of thehyperactive state. “Up to 25%” means, for example, about 1% to about25%. Methods for performing the required measurements are known in theart.

Within the present invention, the disease or condition caused by orassociated with hyperactive cholinergic innervation of muscles includes,but is not limited to, dystonias (e.g., blepharospasm, spasmodictorticollis, limb dystonia, and task-specifc dystonias such as writer'scramps), spasticities (e.g., post-stroke spasticity, spasticity causedby cerebral palsy), paratonia, diskinesias (e.g., tardive diskinesia)focal spasms (e.g., hemifacial spasm), (juvenile) cerebral palsy (e.g.,spastic, dyskinetic or ataxic cerebral palsy), strabismus, pain (e.g.neuropathic pain), wound healing, tremors, tics, and migraine.

The prefilled botulinum toxin container (e.g., a syringe, vial, carpuleor ampoule) of the present invention is particularly useful in thetreatment of dystonia of a muscle. Exemplary dystonias include, but arenot limited to, dystonias selected from the group consisting of (1)cranial dystonia, including blepharospasm and oromandibular dystonia ofthe jaw opening or jaw closing type, (2) cervical dystonia, includingantecollis, retrocollis, laterocollis and torticollis, (3) pharyngealdystonia, (4) laryngeal dystonia, including spasmodic dysphonia, (5)limb dystonia, including arm dystonia such as task specific dystonias(e.g., writer's cramp), leg dystonia, axial dystonia, segmentaldystonia, and (6) other dystonias.

The “hyperactive exocrine gland” to be treated within the context of thepresent invention is not particularly limited and covers any exocrinegland with hyperactivity. It is therefore envisaged that the presentinvention can be applied to the treatment involving any of the glandsmentioned in Sobotta, Johannes, Atlas der Anatomie des Menschen. 22.Auflage. Band 1 and 2, Urban & Fischer, 2005, which is incorporatedherein by reference. Preferably, the hyperactive gland is an autonomicexocrine gland. The botulinum toxin composition is preferably injectedinto or in the vicinity of the hyperactive exocrine gland.

Within the present invention, the hyperactive exocrine gland may beselected from the group consisting of sweat gland, tear gland, salivarygland and mucosal gland. Alternatively, the hyperactive gland may alsobe may be associated with a disease or condition selected from the groupconsisting of Frey syndrome, Crocodile Tears syndrome, axillarhyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis,hyperhidrosis of the head and neck, hyperhidrosis of the body,rhinorrhea, or relative hypersalivation in patients with stroke,Parkinson's disease or amyotrophic lateral sclerosis. In particular, thedisease or condition caused by or associated with hyperactivecholinergic innervation of exocrine glands may include drooling(hypersalivation, sialorrhea) and excessive sweating (hyperhidrosis).

The administration is not limited to any particular administrationregimen, mode, form, dose and interval. As known to those skilled in theart, the administered amount or dose of botulinum toxin depends on themode of application, the type of disease, the patient's weight, age, sexand state of health, and which target tissues are chosen for injection.The botulinum toxin formulation is usually administered locally, e.g.,by subcutaneous or intramuscular injection into or in the vicinity ofthe target tissues (e.g., muscles, skin, exocrine glands).

Furthermore, different muscles, depending on their size, generallyrequire different dosing. A suitable dose may range from 10 to 2000 U,preferably from 50 to 500 U, and more preferably from 100 to 350 U ofbotulinum toxin. For the treatment of exocrine glands, the dose isusually in the range of 10 to 500 U, preferably 20 to 200 U, and morepreferably 30 to 100 U. Such total amounts may be administered on thesame day or on a subsequent day of treatment. For example, during afirst treatment session a first fraction of the dose may beadministered. During one or more treatment sessions, the remainingfraction of the total dose may be administered. Further, the frequencyof application is not particularly limited and suitable administrationintervals may be three months or less (e.g., 4 or 8 weeks) or more thanthree months.

In still another aspect, the present invention relates to the use of theprefilled glass container (e.g., a syringe, vial, carpule or ampoule) ofthe present invention for cosmetic applications, such as for treatingfacial asymmetries and wrinkles and lines of the skin (e.g., faciallines and facial wrinkles), such as upper facial rhytides, platysmabands, glabellar frown lines, nasolabial folds, chin folds, marionettelines, buccal commissures, perioral wrinkles, crow's feet, and jawlines.Preferably, the prefilled botulinum toxin container (e.g., a syringe,vial, carpule or ampoule) of the present invention is used for injectioninto glabellar frown lines, horizontal forehead lines, crow's feet,perioral folds, mental ceases, popply chin, and/or platysmal bands.

The amounts of botulinum toxin administered for cosmetic application areusually in the range of 1 to 5 U, 5 to 10 U, 10 to 20 U or 20 to 50 U.Such total amounts may be administered on the same day or on asubsequent day of treatment. For example, during a first treatmentsession a first fraction of the dose may be administered. This firstfraction is preferably a suboptimal fraction, i.e. a fraction, whichdoes not remove the wrinkles or skin lines completely. During one ormore treatment sessions, the remaining fraction of the total dose may beadministered.

In a yet further aspect, the present invention relates to a method oftreating a disease or condition caused by or associated with hyperactivecholinergic innervation of muscles or exocrine glands in a patient, themethod comprising locally administering an effective amount of botulinumtoxin to a muscle or exocrine gland of the patient using the prefilledglass container (e.g., a syringe, vial, carpule or ampoule) according tothe first aspect of the invention.

The term “effective amount”, as used herein, refers to the amount of abotulinum toxin sufficient to effect beneficial or desired therapeutic,cosmetic or anesthetic result. In the present context, the term “localadministration” within the meaning of the present invention referspreferably to subcutaneous or intramuscular injection into or in thevicinity of the target tissues (e.g., muscles, skin, exocrine glands).The term “patient”, as used herein, generally relates to a humanafflicted with a disease or condition caused by or associated withhyperactive cholinergic innervation of muscles or exocrine glands in apatient, or to a human in need of a cosmetic or anesthetic treatment. Asused herein, “patient” may be interchangeably used with “subject” or“individual”.

The administration is not limited to any particular administrationregimen, mode, form, dose and interval. As used herein, the term “to themuscle or exocrine gland” means that the botulinum toxin may beadministered into, or in vicinity of, one or more muscles or exocrineglands. Usually, the botulinum toxin is administered by localintramuscular injection. With respect to further details regarding theadministration (e.g., regimen, mode, form, dose and interval) and thedisease or conditions to be treated, the same comments apply as thoseset out above in relation to the use of the prefilled glass container(e.g., prefilled botulinum toxin syringe) for cosmetic and therapeuticapplications.

In a still further aspect, the present invention relates to a method forthe cosmetic treatment of the skin, such as for treating wrinkles of theskin and facial asymmetries, the method comprising locally administeringan effective amount of botulinum toxin to a patient by intradermal,subdermal or subcutaneous injection using the prefilled glass syringeaccording to the first aspect of the present invention.

This still further aspect is closely related to other aspects of thepresent invention described above and, thus, all comments, definitionsand explanations given above in relation to these other aspects equallyapply, unless otherwise stated.

The present invention will now be further illustrated by the following,non-limiting examples.

Examples

The following examples demonstrate the superior long-term stability ofan aqueous botulinum toxin formulation in different prefilled syringesystems (in the following referred to as “configurations”) according tothe present invention.

The results obtained for the different syringe configurationssurprisingly show that, contrary to expectation and common belief in theart, an aqueous botulinum toxin formulation stored in a prefilledsyringe system is stable for a prolonged time period (e.g., up to 18months) at standard refrigerator temperature (2-8° C.) and is evenstable when stored for about 9 months at an elevated temperature of 25°C. Furthermore, extrapolation of the measured stability data indicatesthat the prefilled botulinum toxin syringe allows the provision of ashelf life at 2-8° C. of about 24 months and even longer.

Overall, the results obtained show that botulinum toxin can beconveniently used via prefilled syringes. This is an importantcontribution to the management of a wide variety of botulinumtoxin-treated therapeutic and cosmetic indications since botulinum toxinprefilled syringes are safer and more convenient to use for cliniciansand patients compared to conventional lyophilized botulinum toxinproducts, and offer flexibility and excellent shelf life.

Materials & Methods

The liquid botulinum toxin formulation used in the following exampleswas prepared by dissolving 1.0 mg human albumin, 4.7 mg sucrose andincobotulinumtoxinA in 0.9% saline to a concentration of 50 U/ml.

The botulinum toxin solution was then filled into a syringe glass barrelpre-assembled with a Luer-Lock-type closure comprising a Luer-Lockadaptor and a tip cap which, when fitted, contacts the opening of thedistal syringe tip in order to seal the syringe barrel. A plungerstopper was inserted into the proximal end portion of the barrel inorder to close the proximal opening. The resulting prefilled syringe wasthen stored at a temperature of about 5° C., 25° C., and 30° C.

The stability of the botulinum toxin solution was determined initiallyand after a storage time of one month, three months, six months and ninemonths by measuring the remaining toxin potency, the pH value, and thesubvisible particles level.

The potency was determined using a hemidiaphragm assay. The assay isconducted using a murine nerve muscle preparation which is maintained inan organ bath containing 4 ml of medium. The muscle is attached to aforce transducer and electrically stimulated via the phrenic nerveresulting in a isometric contraction force which remains constant formore than 180 min if no toxin is added. Upon introduction of toxin tothe organ bath, the contraction amplitude of the nerve-stimulated musclegradually declines. The contraction amplitude of the diaphragm ismonitored over time. As a read-out, the time at which half the initialcontraction force is reached is determined and referred to as paralysistime. The paralysis time is proportional to the amount of active toxinadded to the preparation.

The pH measurements were performed in accordance with the USPharmacopeia standardized test method USP <791>, which outlines pHmeasurements for a multitude of pharmaceutical product, using a pH meter(Lab 870, Schott Instruments).

Particle measurements were conducted using Micro-Flow Imaging. TheMicro-Flow Imaging measurements were conducted using a DPA-5200 particleanalyzer system (ProteinSimple, Santa Clara, Calif., USA) equipped witha silane coated high-resolution 100 μm flow cell. The samples wereanalyzed undiluted. MFI View System Software (MVSS) version2-R2-6.1.20.1915 was used to perform the measurements, and MFI ViewAnalysis Suite (MVAS) software version 1.3.0.1007 was used to analyzethe samples.

Four different prefilled syringe systems (or “syringe configurations”),which differ from each other by the syringe barrel, tip cap and/orplunger stopper, were examined and are summarized in Table 1.

TABLE 1 Syringe configurations A, B, G, and H studied SYRINGE BARREL TIPCAP PLUNGER STOPPER CO- CO- Product Product Product NF. MP. nameMaterial name Material Name Material A GH¹ RTF ® 1.0 BorosilicateHelvoet ® Styrene- West ® Bromobutyl ml long glass of type 1; FM 27butadiene 4023/50 elastomer Luer Lock inner surface of Greyl rubber Graywith reinforced with syringe glass barrel is compound FluroTec ® aninert mineral with TELC⁴ siliconized by (free from coating and coatedwith “Baked on MBT(2- a FluroTec ® film Siliconization”³; mercaptobenzsterilized by EtO othiazole)) B GH¹ RTF ® 1.0 See Config. A Helvoet ®See Config. A West ® Elastomer ml long FM 27 4023/50G formulation LuerLock greyl NovaPure ® coated with syringe FluroTec ® with barrier filmTELC⁴ G BD² BD Hypak Borosilicate type PRTC Styrene- BD Bromobutyl SCF ™1 I glass; inner FM 27 butadiene Hypak ™ elastomer ml PRTC⁶ surface ofglass Grey⁵ rubber BSCF reinforced with barrel is silicon- compound4023/50 an inert mineral ized using a (free from grey and coated withsilicone oil; MBT) FluroTec ® a FluroTec ® film sterilized by EtO(Weste ® )⁷ (ethylene oxide) H BD² BD Hypak See Config. G PRTC SyntheticBD See Config. G SCF ™ 1 7025/65 isoprene- Hypak ™ ml PRTC⁶ greybromobutyl BSCF (West8)⁵ blend 4023/50 reinforced with grey an inertFluroTec ® mineral (West ® )⁷ ¹= Gerresheimer ²= Becton, Dickinson andCompany ³= Use of Dow Corning ® 365, Dimethicone NF emulsion ⁴= TELC(tamper evident Luerock Closure) ⁵= PRTC (plastic rigid tip cap) ⁶= SCF(sterile, clean and ready-to-fill ⁷= BSCF (bagged sterile, clean andready-to fill; utilizes bagged (BSCF) stoppers)

Results

The results of the stability measurements for configurations A, B, G,and H are shown in Table 2 below.

TABLE 2 Stability in terms of potency STABILITY (toxin potency in %,relative to the initial toxin activity) Time (months) CON- Tempe- t = 0FIG. rature (initial)* 1 3 6 9 12 18 A 2-8° C. 100 102 102 100 107 10988 B 100 109  96 102 102  96 109 G 100 100 100  96  91 102  96 H 100 100114 102 112 100 108 A 25° C. 100  98 102 107  93  89  73 B 100 102 100107  94 107  87 G 100  93 104  96  98  98  75 H 100 108 116 100  90  80 73 A 30° C. 100  84  91  80 — — — B 100 106  93  87 — — — G 100  88  93 84 — — — H 100  92 114  94 — — — *= The initial absolute toxin activityin units ranges from 51 U to 56 U.

The above stability data are, together with an extrapolation to astorage time of 24 months, graphically shown in FIG. 1 (stability at2-8° C.), FIG. 2 (stability at 25° C.), and FIG. 3 (stability at 30°C.). As can be seen from Table 2 and FIGS. 1-3, the maximum measuredloss of biological activity is only 12%, 20%, and 20% for thetemperature conditions 2-8° C. (up to 18 months), 25° C. (up to 12months), and 30° C. (up to 6 months), respectively. Extrapolationsindicate that the loss in biological activity after a storage time of 24months is less than 5% at 2-8° C. for all configurations A, B, G, and H,and less than 10% at 25° C. for configuration B.

Furthermore, the pH measurements revealed that the pH remainedexceptionally stable over a period of up to 18 months. No trend towardshigher or lower values was observed and all measured pH values remainedwithin ±0.5 of the initial pH (see Table 3).

TABLE 3 Stability in terms of pH STABILITY (pH) Time (months) CON-Tempe- t = 0 FIG. rature (initial) 1 3 6 9 12 18 A 2-8° C. 6.8 6.8 7.07.1 6.5 n.d.* 6.8 B 7.0 6.7 6.9 6.9 6.6 n.d. 6.7 G 6.8 6.7 6.7 6.8 6.6n.d. 6.7 H 6.4 6.2 6.6 6.4 6.9 6.6 n.d. A 25° C. 6.8 6.8 7.0 7.0 6.6 6.96.5 B 7.0 6.7 6.9 7.0 6.7 7.0 6.7 G 6.8 6.7 6.7 6.8 6.7 6.8 7.0 H 6.46.3 6.8 6.6 6.7 6.6 n.d. A 30° C. 6.8 6.8 7.0 7.0 — — — B 7.0 6.8 6.97.1 — — — G 6.8 6.8 6.7 6.9 — — — H 6.4 6.4 6.8 6.6 — — — *n.d. = notdetermined Moreover, the particle size measurements by Micro-FlowImaging showed no significant increase in the particle count (see Table4).

TABLE 4 Stability in terms of the subvisible particle count STABILITY(subvisible particle count (equal to or greater than 10 μm)) Time(months) CON- Tempe- t = 0 FIG. rature (initial) 1 3 6 9 12 18 A 2-8° C.27 63 14 27 69 64 162 B 69 80 26 9 64 27 58 G 18 101 48 125 133 83 351 H545 139 59 163 223 n.d. n.d. A 25° C. 27 74 25 43 5 53 127 B 69 22 11 15129 146 143 G 18 176 110 67 105 81 378 H 545 345 227 86 756 327 n.d. A30° C. 27 27 15 53 — — — B 69 42 30 89 — — — G 18 78 89 90 — — — H 545475 150 396 — — —

As can be seen from Table 4, the particle counts stay well below 1000/mland in most cases even below 200/ml. Likewise, particle measurements bymeans of the resonant mass measurement method (using the ARCHIMEDESparticle methodology system; Affinity Biosensors, Santa Barbara, Calif.,USA) and nanoparticle tracking analysis (using a NanoSight LM20 system;NanoSight, Amesbury, UK) revealed no relevant particle counts.

In conclusion, the results presented above show that liquid botulinumtoxin formulations in prefilled syringes are stable for a prolongedperiod at temperatures of 2-8° C. and even at ambient temperatures(e.g., 25° C. to 30° C.). In view of the fact that botulinum toxins areinherently instable, in particular at low toxin concentrations, thisfinding was unexpected. In particular, botulinum toxins are known to behighly heat-labile and highly unstable at alkaline pH. Therefore, giventhe labile nature of botulinum toxins, the finding that botulinum toxinin aqueous solution is highly stable, when stored in prefilled syringes,was highly surprising.

The botulinum toxin prefilled syringe according to the present inventiontherefore offers significant advantages over other ways to deliverbotulinum toxin, including enhancing convenience and ease of handling,reducing medication errors, improving dosing accuracy, minimizing therisk of contamination, improving sterility assurance, and increasingsafety in administration.

1. A method for the cosmetic treatment of the skin, the methodcomprising administering an effective amount of botulinum toxin to apatient using a prefilled glass container, said prefilled glasscontainer comprising an aqueous botulinum toxin formulation, wherein thetoxin activity is not reduced by more than 25%, relative to the initialtoxin activity, upon storage of the prefilled glass container for 12months at 5° C. or 12 months at 25° C. or 6 months at 30° C.
 2. Themethod of claim 1, wherein the effective amount of botulinum toxin islocally administered to a patient.
 3. The method of claim 1, wherein theeffective amount of botulinum toxin is administered to a patient byintramuscular, intradermal, subdermal or subcutaneous injection.
 4. Themethod of claim 3, wherein the effective amount of botulinum toxin isadministered to a patient by intramuscular injection.
 5. The method ofclaim 3, wherein the effective amount of botulinum toxin is administeredto a patient by intradermal, subdermal or subcutaneous injection.
 6. Themethod of claim 1, comprising treating wrinkles and lines of the skinand facial asymmetries.
 7. The method of claim 6, wherein the wrinklesand lines of the skin comprise upper facial rhytides, platysmal bands,glabellar frown lines, horizontal forehead lines, nasolabial folds, chinfolds, mental ceases, marionette lines, buccal commissures, perioralwrinkles, crow's feet, and jawlines.
 8. The method of claim 1, whereinthe botulinum toxin is administered in a total amount of 1 to 50 U. 9.The prefilled glass container of claim 1, wherein the toxin activity isnot reduced by more than 15%, relative to the initial toxin activity,upon storage of the prefilled glass container for 12 months at 5° C. or12 months at 25° C. or 6 months at 30° C.
 10. The method of claim 1,wherein the number of subvisible particles of equal to or greater than10 μm is below 1000/ml during storage of the prefilled glass containerfor 6 to 24 months at 2° C. to 30° C.
 11. The method of claim 1, whereinthe pH value of the aqueous botulinum toxin formulation is not increasedor decreased by more than 10%, relative to the initial pH value, uponstorage of the prefilled glass container for 6 to 12 months at 5° C. or25° C. or 30° C., or wherein the pH of the aqueous botulinum toxinformulation during storage of the prefilled glass container ismaintained in the range of 6.0 to 7.5, or both.
 12. The method of claim1, wherein the botulinum toxin in the aqueous formulation in theprefilled glass container is present at a concentration of 10 U/ml to1000 U/ml.
 13. The method of claim 1, wherein the aqueous botulinumtoxin formulation in the prefilled glass container does not contain abuffer.
 14. The method of claim 1, wherein the prefilled glass containeris (i) a syringe, (ii) a vial, (iii) a carpule, or (iv) an ampoule. 15.The method of claim 14, wherein the prefilled glass container is in theform of a prefilled glass syringe, comprising: (a) a syringe barrel madeof glass including a proximal end and a distal end, and a cylindricalwall extending therebetween and defining a barrel lumen, the syringebarrel having a distally projecting tip with a fluid passage extendingtherethrough and communicating with the barrel lumen, wherein thecylindrical wall has an interior surface optionally coated with abarrier layer, (b) a capping device having an outlet engaging portionsealingly engaging and closing the distal open outlet end of thesyringe, wherein the outlet engaging portion is made of an elastomericmaterial that optionally has a coating on its surface, and (c) a plungerrod assembly which extends into the proximal end of the syringe barreland includes a plunger stopper in sliding fluid-tight engagement withthe cylindrical wall of the barrel lumen, wherein the plunger stopper ismade of an elastomeric material, which optionally has a coating on atleast a portion of the plunger stopper that contacts the aqueousbotulinum toxin formulation during storage and/or injection.
 16. Themethod of claim 15, wherein the prefilled glass container is in the formof a prefilled glass syringe, and wherein the elastomeric material ofthe outlet engaging portion and/or the plunger stopper is selected fromisoprene rubber (IS), butadiene rubber (BR), butyl rubber, halogenatedbutyl rubbers, styrene-butadiene rubber, and mixtures thereof, orwherein the optional coating on the outlet engaging portion and/or theplunger stopper is a crosslinked silicone coating or a fluoropolymercoating, or wherein the elastomeric material of the outlet engagingportion and/or the plunger stopper is selected from isoprene rubber(IS), butadiene rubber (BR), butyl rubber, halogenated butyl rubbers,styrene-butadiene rubber, and mixtures thereof, and the optional coatingon the outlet engaging portion and/or the plunger stopper is acrosslinked silicone coating or a fluoropolymer coating.
 17. The methodof claim 15, wherein the prefilled glass container is in the form of aprefilled glass syringe, and wherein the barrier layer of the syringebarrel is a silicone-free layer or a silicone layer.